Abstract

We examined the genomewide transcriptional responses of Escherichia coli treated with nitrosylated glutathione or the nitric oxide (NO)-generator acidified sodium nitrite (NaNO2) during aerobic growth. These assays showed that NorR, a homolog of NO-responsive transcription factors in Ralstonia eutrophus, and Fur, the global repressor of ferric ion uptake, are major regulators of the response to reactive nitrogen species. In contrast, SoxR and OxyR, regulators of the E. coli defenses against superoxide-generating compounds and hydrogen peroxide, respectively, have minor roles. Moreover, additional regulators of the E. coli response to reactive nitrogen species remain to be identified because several of the induced genes were regulated normally in norR, fur, soxRS, and oxyR mutant strains. We propose that the E. coli transcriptional response to reactive nitrogen species is a composite response mediated by the modification of multiple transcription factors containing iron or redox-active cysteines, some specifically designed to sense NO and its derivatives and others that are collaterally activated by the reactive nitrogen species.

NO gas is generated during the combustion of nitrogenous compounds and the biological decay of organic matter, and as a byproduct of denitrification reactions carried out by microbes. NO is also produced directly by NO synthases in animals, plants, and bacteria (reviewed in ref. 1). At low concentrations, NO functions as a signaling molecule, whereas at high concentrations, NO can be a general poison (reviewed in refs. 2–4). At elevated levels, the soluble gas reacts with heme centers and labile 4Fe–4S clusters and thus inhibits terminal oxidases and aerobic respiration (reviewed in refs. 5 and 6). NO also can react with superoxide (O2-·) to generate peroxynitrite (OONO-), which can react with other molecules and decompose to the highly reactive hydroxyl (·OH) and nitrogen dioxide (NO2·) radicals. In addition, NO-derived species can react with small molecule and protein thiols, thereby disrupting protein activity as well as depleting the reduced glutathione pool and generating nitrosylated glutathione (GSNO), which in turn can modify proteins.

Because of the prominent role of reactive nitrogen species in macrophage killing of bacteria, several activities that scavenge or detoxify NO, OONO-, or GSNO or repair damage caused by these compounds have been characterized in Salmonella enterica serovar Typhimurium and Escherichia coli. For example, it has been proposed that homocysteine reacts with nitrosylating compounds and acts as an NO antagonist because S. enterica strains carrying insertions in metL encoding aspartokinase II–homoserine dehydrogenase II are hypersensitive to S-nitrosothiol (7). The most prominent detoxifying entities that have been identified are the NO dioxygenase, NO denitrosylase, and NO reductase activities associated with the hmpA-encoded flavohemoglobin and the NO reductase activity associated with the norVW-encoded flavorubedoxin and flavorubedoxin reductase (8–16). Other detoxifying activities include NO reductase activity contributed by nfrA-encoded periplasmic cytochrome c nitrite reductase (17), and GSNO reductase activity associated with the adhC-encoded alcohol-acetaldehyde dehydrogenase (18). The AhpC subunit of the alkylhydroperoxide reductase also has been shown to catalyze the conversion of OONO- to NO2 (19). Finally, the peptide methionine sulfoxide reductase encoded by msrA has been proposed to repair intracellular methionine residues damaged by OONO- (20).

A number of E. coli and S. enterica transcriptional regulators have been implicated in modulating gene expression in response to reactive nitrogen species. The E. coli norR (ygaA) gene encodes a homolog of the NO-modulated NorR1 and NorR2 regulators of Ralstonia eutrophus (21), and is required for the induction of norV–lacZ fusions (15, 16, 22). In E. coli, the NO-induction of the hmpA gene was reported to be dependent on MetR, a transcriptional regulator of the methionine biosynthetic pathway, under aerobic conditions (23) and on FNR, an oxygen-responsive regulator, under anaerobic conditions (24). In S. enterica, the aerobic induction of hmpA was reported to depend on Fur, the ferric iron repressor (25).

Reactive nitrogen and reactive oxygen species share chemical properties, and two regulators of the E. coli responses to oxidative stress can be modified by reactive nitrogen species. The iron–sulfur cluster-containing SoxR protein initially was identified as the sensor of the stress generated by O2-·-generating compounds, but subsequent studies revealed that SoxR also can be activated by NO (26). Similarly, the OxyR transcription factor, discovered as the primary regulator of the response to hydrogen peroxide (H2O2), later was reported to activate target genes on exposure to S-nitrosylated cysteine (27). We have shown that OxyR activation by H2O2 is caused by disulfide bond formation between residues Cys-199 and Cys-208 (28, 29). However, others have reported that Cys-199 is modified to S-NO, S-OH, and S-SG and have proposed that these modifications of OxyR elicit different responses at increasing concentrations of GSNO (30). The relative importance the OxyR and SoxR regulons in the response to reactive nitrogen species has not been evaluated on a genomewide level.

To define the global E. coli transcriptional response to reactive nitrogen species, we carried out microarray analyses. The recently developed transcriptional profiling technologies allow unbiased, genomewide surveys of an organism's response and have been very successful in characterizing E. coli gene expression changes under many different growth and stress conditions (reviewed in ref. 31). We also set out to evaluate the relative contributions of each of the implicated transcriptional regulators by examining the global gene expression patterns as well as the expression of specific genes in mutant strain backgrounds.

Materials and Methods

Strains. The sequences of primers used in this study are given in the supporting information, which is published on the PNAS web site. E. coli MG1655 was the parental strain used in this study. The MG1655 ΔoxyR::kan derivative (GSO77) was described (32). The Δfur::kan (33) and ΔsoxRS–zjc2205 zjc2204::Tn10kan (34) mutant alleles were moved into MG1655 by P1 transduction to generate GSO82 and GSO83, respectively. The npt (kanamycin gene) replacements of norR and metR were constructed as described (35). PCR fragments obtained by amplifying the kanamycin resistance cassette from pKD4 (36) with primers carrying sequences flanking norR and metR were purified by using the Gel Extraction kit (Qiagen, Valencia, CA) and transformed into DY330 (35). Recombinants were selected on the basis of kanamycin resistance and confirmed by colony PCR (using one primer homologous to the 5′ flanking sequence of the targeted gene and a second primer homologous to the inserted kanamycin resistance gene). The ΔnorR::kan and ΔmetR::kan mutant alleles then were moved into MG1655 by P1 transduction to generate GSO86 and GSO87, respectively.

RNA Isolation. For the aerobic samples, cultures were grown at 37°C with shaking in LB-rich medium adjusted to pH 6 with HCl. Overnight cultures were diluted 1:100 and grown to OD600 = 0.1, 0.4, or 1.0 under the same conditions. For the anaerobic samples, cultures were grown at 37°C with stirring in an anaerobic glove box (Coy Laboratory Products, Grass Lake, MI) in LB medium buffered at pH 6 with 100 mM Mes and supplemented with 0.3% glucose. Overnight cultures were diluted 1:100 and grown to OD600 = 0.4 under the same conditions. Aliquots (15 or 30 ml) of the cultures at the indicated stages of growth were left untreated or exposed to the indicated concentrations of H2O2, acidified NaNO2, or GSNO. After the indicated times, total RNA was isolated and purified by using TRIZOL reagent (Invitrogen) according to the manufacturer's instructions.

DNA Microarray Analysis. Fabrication of the E. coli DNA microarray and procedures for cDNA labeling, hybridization, and array quantification were carried out as detailed (38, 39). Briefly, each RNA sample was used as a template for two cDNA syntheses, each with separate incorporation of Cy3- and Cy5-labeled nucleotides. The reciprocal pairs of differentially labeled, untreated, and treated cDNA samples were hybridized to glass slides printed in duplicate with 4,169 E. coli ORFs. Thus, the expression for each gene was measured four times for each RNA sample. The numbers were averaged, and the treated sample/untreated sample ratio was calculated for each ORF. The complete microarray data sets are available in the supporting information.

Primer Extension and Northern Blot Assays. RNA samples were subjected to primer extension analysis as described (40), using avian myeloblastosis virus reverse transcriptase (Life Sciences, St. Petersburg, FL) and primers specific to the indicated genes. The extension products were separated on 8% sequencing gels. Northern blot analyses were carried out as described (41).

Results and Discussion

Global Responses to GSNO and NaNO2. We examined the transcriptional profile of E. coli cells exposed to two commonly used generators of reactive nitrogen species: GSNO, a representative nitrosothiol, and acidified NaNO2, a NO producer. Wild-type cells (MG1655) grown aerobically to exponential phase in LB-rich medium adjusted to pH 6 were left untreated or exposed to GSNO or NaNO2 each at 0.1 and 1 mM. After 5 min, total RNA was isolated from the untreated and treated cultures. Neither concentration resulted in a loss of viability during the time frame of the experiments, and primer extension assays confirmed that the expression of hmpA, a gene previously shown to be induced by exposure to reactive nitrogen species (23–25, 42), was elevated in the samples from the GSNO- or NaNO2-treated cells. Fluorescently labeled cDNAs derived from these total RNA samples were used to probe glass slide arrays printed with 4,169 E. coli ORFs.

For all four conditions, RNA was isolated from two independent sets of untreated and treated cultures. RNA also was isolated from a separate set of wild-type, ΔnorR, Δfur, and ΔoxyR cells treated with 1 mM GSNO. The complete data sets for all arrays are given in the supporting information. For each of the treatments, only limited numbers of genes were induced ≥5-fold (36, 44, 47, and 49 genes for 0.1 and 1 mM GSNO and 0.1 and 1 mM NaNO2, respectively). The 34 genes whose transcripts showed ≥5-fold accumulation in the samples treated with 1 mM GSNO and the samples treated with 1 mM NaNO2 are listed in Table 1. A similarly small set of 48 genes was found to be induced in a recent microarray analysis of Mycobacterium tuberculosis exposed to nontoxic concentrations of NO (43). Only 5 and 7 genes were repressed ≥2-fold by 0.1 mM concentrations of GSNO and NaNO2, respectively, and several hundred genes were repressed ≥2-fold by 1 mM concentrations.

Known Defense Activities Induced by GSNO and NaNO2. As described above, a number of proteins have been implicated in protecting cells against reactive nitrogen species. Among the corresponding genes, three (norV, norW, and hmpA) were induced strongly (>30-fold) by both concentrations of GSNO and NaNO2 (Table 1). These results are consistent with the conclusion (15) that the NO dioxygenase and NO reductase activities encoded by norV, norW, and hmpA play critical roles in protecting E. coli cells against the toxic effects of NO. The transcript levels for other postulated defense genes such as metL, nrfA, adhC, ahpC, and msrA showed no or only minimal (<2-fold) induction under our culture conditions. It may be that basal levels of some of the corresponding proteins suffice to protect against the reactive nitrogen species. Alternatively, these defense activities may not be critical during aerobic growth in rich medium and instead are induced and protective under other culture conditions. Several genes of unknown function (such as ybaE and ychH) were induced ≥5-fold by both GSNO and NaNO2 (Table 1), suggesting that these genes may be important to the E. coli response to reactive nitrogen species. Further characterization of induced genes with unidentified function will likely reveal additional activities that protect against damage caused by reactive nitrogen species.

Major Contributions of the NorR and Fur Transcription Factors. The expression of the two most strongly regulated genes, norV and norW, has been shown to be regulated by NorR by assays of lacZ operon fusions (15, 16, 22). NorR regulation of these two genes was confirmed by microarray analysis of total RNA isolated from ΔnorR mutant with and without exposure to 1 mM GSNO; induction of norV and norW was abolished in the ΔnorR mutant strain. The induction of other genes, such as ybiJ, was also eliminated, suggesting that these genes may be additional NorR targets. Primer extension assays of norV mRNA levels in strains carrying mutations in all of the transcription factors implicated in the response to reactive nitrogen species under aerobic conditions showed that only the NorR regulator is required for the GSNO and NaNO2 induction of norV (Fig. 1).

Primer extension assays of norV, fes, ydiE, grxA, ahpC, katG, sodA, uspA, hmpA, and ygbA expression in wild-type and isogenic regulatory mutants. The parent MG1655 and Δfur, ΔnorR, ΔoxyR, ΔsoxRS, and ΔmetR mutant strains were grown to OD600 = 0.4–0.6 in LB medium, pH 6. Cultures were split, and aliquots were left untreated or treated with 1 mM H2O2, NaNO2, or GSNO. After 5 min, total RNA was isolated from each untreated and treated culture. All assays were carried out by using 5 μg of the same RNA preparation.

Four of the genes induced ≥5-fold by 1 mM GSNO and NaNO2 (fes, nrdH, sufA, and fhuF) and 13 of the genes induced ≥5-fold by the 0.1 mM concentrations are members of the Fur regulon (44). In microarrays of Δfur mutants treated with 1 mM GSNO, the induction ratios of the Fur-repressed genes were all reduced to <2-fold, indicating that GSNO induction of these genes occurs via the Fur regulator. The primer extension assays also showed that the mRNA levels for fes as well as ydiE, another known Fur target gene, in untreated Δfur mutants were similar to the levels in wild-type cells treated with 1 mM H2O2, GSNO, or NaNO2 (Fig. 1). These results suggest that elevated expression of these genes in response to H2O2, GSNO, or NaNO2 is due to relief from Fur repression.

To further assess the roles of the NorR and Fur transcription factors in protecting cells against deleterious effects of reactive nitrogen species, we also examined the growth of wild-type and mutant strains treated with 3 mM GSNO or 2 mM NaNO2 (shown in supporting information). The ΔnorR mutants recovered quickly from the exposure to either GSNO or NaNO2 and did not show a substantial growth delay compared to the parental strain. We suggest this lack of sensitivity may be due to some redundancy between the norVW-encoded NO reductase and other enzymes such as the hmpA-encoded NO reductase. The Δfur mutant exhibited severe growth delays on both treatments. This finding that Δfur mutants, in which Fur target genes are constitutively derepressed, were hypersensitive to GSNO and NaNO2 is consistent with a prominent Fur role in the E. coli response to reactive nitrogen species, but seems contradictory to the observation that Fur target genes are derepressed on GSNO treatment. However, Fur is an extremely abundant protein of >10,000 molecules per cell (45) and may perform functions other than its role as a transcriptional repressor. In addition, the benefits of temporary induction of Fur target genes may not be observed on constitutive derepression of these genes.

Minor Contributions of the SoxR and OxyR Transcription Factors. The expression of soxS, whose transcription is controlled directly by SoxR was induced ≥5-fold by both GSNO and NaNO2. However, with the exceptions of fpr (induced 3.5- and 3.0-fold by GSNO and NaNO2, respectively) and sodA (induced 2.7- and 2.8-fold), none of the other known members of the SoxRS regulon were induced >2-fold. The induction of sodA is through SoxRS because the primer extension assays in Fig. 1 showed that sodA induction by H2O2, GSNO, or NaNO2 was reduced in the ΔsoxRS mutant strain, consistent with previous findings (26).

The OxyR transcription factor has been described as a sensor of nitrosative stress in E. coli (27, 30). However, the only OxyR target genes, sufA and fhuF, showing ≥5-fold induction by GSNO or NaNO2, are known to be regulated by both Fur and OxyR. Although one other target gene, grxA, was induced 4-fold, no other known target was elevated >2-fold. To further explore the OxyR role in the responses to GSNO or NaNO2 and to compare these to the OxyR-mediated response to H2O2, we also assayed the expression of three target genes by primer extension (Fig. 1). All three genes were strongly induced by H2O2, but showed differences in induction by GSNO and NaNO2; grxA was induced moderately by both compounds via OxyR and ahpC showed slight induction, whereas there was no detectable induction of katG. The katG gene was shown to be activated by nitrosylated OxyR in vitro (30), and a plasmid-borne katG–lacZ fusion was reported to be induced by 1 mM S-nitrosocysteine in vivo (27). However, in our measures of katG mRNA levels produced from the normal location in a wild-type strain, we do not detect any induction by reactive nitrogen species under any of the conditions that we have tried, including exposure to 0.1, 1, and 5 mM GSNO, NaNO2, sodium nitrosoprusside, or diethylenetriamine (DETA) NONOate over a 90-min time course (data not shown). Given that we see strongly elevated mRNA levels for other genes previously reported to be NO-regulated in the identical total RNA sample, the lack of katG induction cannot be attributed to a use of inactive NO generators. The ΔsoxRS and ΔoxyR mutants also both showed wild-type sensitivity to 3 mM GSNO and 2 mM NaNO2.

These results indicate that SoxR and OxyR do not serve as primary regulators of the E. coli response to reactive nitrogen species during aerobic growth. Although the redox-active Cys-199 residue in OxyR probably can undergo modification to S-NO, this form of the protein is at best capable of limited activation of a subset of promoters. Thus, we propose that the E. coli response to reactive nitrogen species is brought about by the modification of some transcription factors, such as NorR, that control the expression of NO detoxifying activities and are dedicated to sensing reactive nitrogen species, and other redox-active transcription factors such as Fur, SoxR, and OxyR, where the reaction with reactive nitrogen species is incidental. In this context, the E. coli NorR protein should be favored as a model for examining effects of reactive nitrogen species on transcription factor activity.

Additional Transcriptional Regulators of the Responses to GSNO and NaNO2. The microarray analysis revealed that the induction of several of the genes was unaffected in the Δfur, ΔnorR, and ΔoxyR mutant strains. We further examined expression of three of these genes by primer extension assays (Fig. 1). These experiments showed that uspA, hmpA, and ygbA genes had wild-type induction by GSNO and NaNO2 in the Δfur, ΔnorR, ΔoxyR, ΔsoxRS, and ΔmetR backgrounds. Thus, additional regulators must exist whose activities are modulated, directly or indirectly, by reactive nitrogen species. Our finding that induction of hmpA by GSNO under aerobic conditions did not require MetR contradicts a previous study of an hmpA–lacZ fusion (23), but possibly can be explained by differences in strain backgrounds or growth conditions.

Oscillatory Expression of norV. To determine whether the SoxRS and OxyR regulators play more important roles at different time points or under other conditions, we also assayed the dose dependence (Fig. 2A) and time course of induction (Fig. 2B) for representative target genes. The expression profiles showed differences that are likely to have consequences for the E. coli response to reactive nitrogen species. The norV, ydiE, soxS, grxA, and hmpA genes all were maximally induced by 1 mM GSNO and NaNO2, whereas ygbA was induced equally by 0.1 and 1 mM concentrations. The norV, ydiE, and grxA transcript levels also were slightly induced by 0.1 mM NaNO2, and hmpA and ygbA even showed induction by 0.01 mM concentrations of both GSNO and NaNO2 indicating exquisite sensitivity to these reactive nitrogen species. The elevated levels of the ygbA mRNA persisted for >90 min. In contrast, the expression of ydiE, soxS, and grxA, targets of Fur, SoxRS, and OxyR, respectively, was maximal at 5 min and then returned to pretreatment levels within 15 min. Surprisingly, the two genes encoding known NO-detoxification activities, norV and hmpA, showed two peaks of induction, one peak at 5 min and another at 90 min.

Primer extension assays of norV, ydiE, soxS, grxA, hmpA, and ygbA expression in wild-type cells treated with 0.01, 0.1, 1, and 5 mM GSNO or NaNO2 for 5 min (A) or with 1 mM GSNO or NaNO2 for 5, 10, 20, 30, 40, 50, 60, or 90 min (B). MG1655 cells were grown to OD600 = 0.4 in LB medium, pH 6. Aliquots of the cultures were left untreated or treated with the indicated concentrations of GSNO or NaNO2 and harvested after the indicated times. Total RNA was isolated from 15 ml of each untreated and treated culture, and 5 μg of each RNA sample was used in each primer extension reaction by using primers to the indicated genes.

The apparent oscillation in norV mRNA levels was examined in more detail by monitoring norV expression for 3 h in cells exposed to 1 mM NaNO2 at different stages of aerobic growth (Fig. 3). Multiple peaks of induction were observed for all three aerobic cultures. Interestingly, however, the period between the peaks varied among the cultures; the period for the early exponential phase culture was ≈45 min, the period for the mid-exponential phase culture was ≈75 min, and the period for the early stationary phase culture was ≈90 min. Simultaneous measurements of the OD600 and pH of the cultures showed that all cultures continued to grow and that the pH of the cultures did not change significantly (shown in supporting information), and Northern blots probed for a control RNA confirmed that equal amounts of total RNA were isolated (Fig. 3). For anaerobically grown cells treated with 1 mM NaNO2 at mid-exponential phase, norV mRNA levels remained high for the duration of the experiment and no oscillation was observed, suggesting that oxygen is required for the undulating expression pattern.

Primer extension assays of norV expression in aerobic and anaerobic wild-type cells treated with 1 mM NaNO2. MG1655 cells were grown aerobically to OD600 = 0.1, 0.4, and 1.0 or anaerobically to OD600 = 0.4 and treated with 1 mM NaNO2 for 0, 5, 20, 60, 90, 120, 150, or 180 min. Total RNA was isolated from 15–30 ml of each untreated and treated culture; 5 μg of each RNA sample was used in each primer extension reaction using the norV-specific primer, and 1 μg of each RNA sample was used in Northern analysis for the 6S RNA.

Various groups have constructed synthetic genetic circuits that display oscillatory behavior (46–48). In one case, the oscillator consisted of three genes encoding repressors linked in a daisy chain; in the second case, oscillatory expression was achieved by the differential degradation and synthesis of an inhibitory protein; in the third case, the oscillator was composed of an activator and repressor that cross-regulated each other's expression. Given that only a limited number of natural genetic circuits have been found to exhibit oscillatory behavior, it is noteworthy that some human fibroblast genes induced by the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) also show biphasic expression (49). Further characterization of the regulatory mechanisms underlying the oscillations in norV mRNA levels should provide insights into the metabolism of reactive nitrogen species.

Regulation by Multiple Redox-Active Transcription Factors. Our studies of the global transcription response of E. coli cells treated with GSNO and NaNO2 showed that the E. coli response to reactive nitrogen species during aerobic growth in rich media is a composite response in which NorR and Fur have major roles, SoxR and OxyR have minor roles, and additional regulators remain to be identified. It is interesting to note that NorR, Fur, SoxR, and OxyR all appear to contain redox-active cysteines and, in some cases, iron. The mechanism of NorR activation remains to be elucidated, but the E. coli NorR protein has seven cysteines, three of which are conserved in R. eutropha NorR1 and NorR2 and two of which are not conserved but are found in a CXXC motif characteristic of redox-active cysteines. For the E. coli Fur repressor whose activity is regulated by cellular iron levels, NO was found to react with the bound iron to form an iron–Fur–NO complex and thereby abolish the DNA binding activity of this repressor (50). NO also has been shown to react directly with the 2Fe–2S centers of the E. coli SoxR dimer to generate a dinitrosyl–iron–dithiol form of SoxR (51), and as described above, GSNO can react with the redox-active C199 residue in OxyR. We suggest that NorR, and perhaps the unidentified regulators, were evolved to sense reactive nitrogen species, whereas Fur, SoxR, and OxyR were evolved to sense iron and reactive oxygen species and are collaterally activated by reactive nitrogen species.

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